环介导技术在流感检测中的应用与研究

The application and study of LAMP technique in detection for influenza virus

目的建立并应用RT-LAMP检测新甲型H1N1、季节性H1N1和季节性H3N2亚型流感病毒及乙型流感病毒。方法通过在线软件Primer Explorer V5设计针对新甲型H1N1、季节性H1N1和季节性H3N2亚型及乙型流感病毒基因的保守区引物,建立RT-LAMP检测方法,对2012年-2013年采集的疑似咽拭子标本采用real-time PCR和RTLAMP法检测。结果 615份标本中,real-time PCR法检出295份阳性,RT-LAMP法检出294份阳性,2种方法的检测结果差异无统计学意义(P>0.05)。2种方法所建立的季节性H1N1、季节性H3N2、新甲型H1N1、乙型的检测体系只与相应亚型流感病毒呈阳性反应,而对禽流感病毒H5、禽流感病毒H9、RSV、RV、EV71扩增结果为阴性。RT-LAMP检测方法具有良好的敏感性和特异性。结论 RT-LAMP法使用水浴锅进行等温扩增,结果在可见光或紫外灯下直接肉眼观察。RT-LAMP检测方法具有高度的敏感性、准确性、高特异性,而且简单、快速,比real-time PCR更适合应用于基层疾病预防控制中心和医院。

Objective To establish and apply the methods of RT - LAMP assay for the detection of human seasonal influenza HtN1, H3N2, B and novel influenza H1N1. Methods The specific primers of the conserved regions of influenza virus gene for human seasonal influenza H1N1, H3N2, B and novel influenza H1N1 were designed by online Primer Explorer V5 and com- posed to establish the RT - LAMP assay for detection of influenza virus. The throat swab specimens for patients who met the defi- nition of influenza from 2012 to 2013 was collected and test by real - time PCR and RT - LAMP. Results 295 specimens were determined as positive by real - time PCR and 294 by RT - LAMP in 615 specimens. There was no statistical difference in the specificity of 2 methods （P 〉 0. 05 ）. The results showed that the two methods appeared positive for corresponding human seasonal influenza H1N1, H3N2, B and novel influenza H1N1. The assay could not amplify any genes of H9 AIV, H5 AIV, RSV, RV and EV71. RT - LAMP showed fine specificity and high sensitivity. Conclusion RT - LAMP assay was performed in water ov- en and the results could be directly determined by eyes under visible light or UV lamp. RT - LAMP assay for detection of human influenza virus is highly sensitive, accurate, high specific, simple and rapid, which is better than real - time PCR in primary diseases control and prevention centers and hospitals.